Molecular diagnostic methods have become a commonplace tool not only in human genetics, but in microbial genomics, fungal genomics, and mammalian genomics. This is due to advances in DNA/RNA extraction techniques, sequencing methods, amplification techniques, simpler detection methods and the introduction of semiautomated instruments.
The measurement of genetic transcription and gene production by real-time amplification methods are increasingly being used to investigate microbes and biological organisms. However, traditional protocols typically require laboratory analysis and a separation between sample collection and analysis.
In marine waters and other difficult matrices, sample preparation and extraction is key to successful noise and interference reduction and signal enhancement. For capture and purification of target nucleic acids from natural samples it is essential to utilize an efficient binding material that has high selectivity. A material of initial choice is solid phase specific or non-specific binding surface materials. The material can be suitable for irreversible binding of single stranded DNA and RNA and permits capture, isolation, and potential amplification of the captured nucleic acids directly on the solid phase using any amplification strategy. The material can be derivatized with specific oligonucleotides for added capture specificity where few copies of targets are present in complex matrix. The material also provides archiving capability in case samples need to be brought back to the lab for further analysis.
Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization, thus signaling the presence of nucleic acids in solutions and monitoring the synthesis of amplified products in amplification reactions. Employing existing technology, it is possible to engineer different probe beacons having different colored wavelength emissions so that multiple targets can be simultaneously detected allowing for multiplexed assay and internal quality controls. Also since the probes may be engineered differently, probes can be made for any of the manifold biospecies existing in nature.
Devices and reaction vessels or other containers for the lysis of cells, viruses, macromoloecules, particles, or other components within a sample are disclosed in U.S. Pat. Nos. 5,958,349; 6,369,893; and 6,431,476 and U.S. Patent Application Publication Nos. US 2002/0039783, US 2002/0045246, US 2002/0109844, US 2002/0168299, and US 2002/0187547, (which are hereby incorporated by reference in their entirety).
U.S. Pat. No. 6,586,234 (which is hereby incorporated by reference in its entirety) discloses a device for separating an analyte, including nucleic acids, from a fluid sample comprises a cartridge incorporating a flow-through microfluidic chip. As disclosed therein, the cartridge may optionally include a lysing region for lysing sample components (e.g., cells spores, or microorganisms), a waste chamber for storing waste fluid, and reaction or detection chambers for amplifying or detecting the analyte. According to the '234 patent, this technique allows the entire processing facility to be small, yet capable of processing relatively large fluid samples (e.g., 0.1 to 10 mL).
Nucleic acid amplification technology is well known, and is discussed in documents including U.S. Pat. Nos. 4,683,195; 4,683,202; 5,130,238; 4,876,187; 5,030,557; 5,399,491; 5,409,818; 5,485,184; 5,409,818; 5,554,517; 5,437,990; 5,554,516; 6,312,929; 6,534,645; and 6,586,234 and U.S. Patent Application Publication Nos. US 2002/0034745, US 2002/0058282, US 2002/0031768, and US 2002/0034746 (each of which are hereby incorporated by reference in their entirety). It is well known that methods such as those described in these patents permit the amplification and detection of nucleic acids without requiring cloning, and are responsible for the most sensitive assays for nucleic acid sequences. It is further known that computer programs may be employed to provide a highly reproducible quantitative analysis of a nucleic acid amplification reaction.
The use of flow-through microfluidic chips to separate an analyte from a fluid sample was disclosed in U.S. Pat. No. 6,664,104 (which is hereby incorporated by reference in its entirety), which teaches the combination of a cartridge containing such a chip with an optional lysing region, waste chamber for storing waste fluid, and reaction or detection chambers for amplifying or detecting the analyte.
A reaction vessel in combination with a temperature control system for performing heat-exchanging chemical reactions such as nucleic acid amplification is disclosed by U.S. Pat. No. 6,403,037 (which is hereby incorporated by reference in its entirety). A reaction vessel for holding a sample for a heat-exchanging chemical process such as polymerase chain reaction is taught by U.S. Pat. No. 5,958,349 (which is also hereby incorporated by reference in its entirety).
Chips with non-planar microstructures have also been used to manipulate materials including particles, cells, macromoloecules, proteins, nucleic acids, and other moieties in fluid samples according to U.S. Pat. No. 6,368,871 (which is hereby incorporated by reference in its entirety).
Other systems for the manipulation and processing of fluid samples is disclosed by U.S. Pat. Nos. 6,374,684 and 6,440,725 and U.S. Patent Application Publications US 2002/0019060, US 2002/0025576, US 2002/0012612, and US 2002/0175079 (which are hereby incorporated by reference in their entirety).
The foregoing known technologies provide a means for the analysis of an analyte in a fluid or gaseous medium, but do so only in a laboratory setting, with the need for a human operator to control the detection and analysis processes. In many potential applications, particularly in the fields of environmental research, biochemical warfare, hazardous matter situations, and space research, it would be advantageous to develop compact, self-contained analyzers that could autonomously detect and analyze chemical, biochemical, radiologic, or biogenetic expression activity under deployed field conditions to perform specimen collection and concurrently provide real-time analysis of those specimens.
More specifically, the need exists for a system to detect target microorganisms and larger biological organisms in both the marine and fresh water environments, as well as sources of drinking water or in any industrial process that may have microbial content concerns. It would be additionally desirable for such a system to provide for the automatic and autonomous detection and analysis of target gene sequences from desired organisms and further provide for the transmission of the data generated to remote data collection centers in near real time. Sample amplification in such a system would also be desirable, and could be performed using any of the existing nucleic acid amplification methods currently commercially available specifically, PCR—Polymerase Chain Reaction, RT-PCR—Reverse Transcription PCR, and NASBA—Nucleic Acid Sequence Based Amplification (NASBA) method.